Oxidative Stress-Associated Protein Tyrosine Kinases and Phosphatases in Fanconi Anemia

Abstract

Significance: Fanconi anemia (FA) is a genetic disorder featuring chromosomal instability, developmental defects, progressive bone marrow failure, and predisposition to cancer. Besides the predominant role in DNA damage response and/or repair, many studies have linked FA proteins to oxidative stress. Oxidative stress, defined as imbalance in pro-oxidant and antioxidant homeostasis, has been considered to contribute to disease development, including FA. Recent Advances: A variety of signaling pathways may be influenced by oxidative stress, particularly the equilibrium between protein kinases and phosphatases, consequently leading to an aberrant phosphorylation state of cellular proteins. Dysfunction of kinases/phosphatases has been implicated in the pathophysiology of human diseases. In FA, evidence is emerging that links abnormal phosphorylation/de-phosphorylation of signaling molecules to clinical complications and malformations. Critical Issues: In this study, we review the recent findings on the oxidative stress-related kinases and phosphatases, particularly tyrosine phosphatases in FA. Future Directions: Understanding the role of oxidative stress-related kinases and phosphatases in FA may provide unique and generic possibilities for the future development of therapeutic strategies by targeting the dysregulated protein kinases and phosphatases in a clinical setting. Antioxid. Redox Signal. 20, 2290–2301.

Introduction

Fanconi anemia (FA) is a rare genetic disease associated with chromosomal instability, bone marrow failure, developmental defects, cancer risk, and a broad array of clinical complications and malformations (20, 78, 129). To date, 15 FA complementation groups and the corresponding genes have been identified, including FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, and P, which may function through the FA-BRCA pathway (20, 22, 53, 55, 78, 108, 129). Among them, eight of the FA proteins (composed of FANCA, B, C, E, F, G, L, and M) form a nuclear complex and act as a ubiquitin ligase in response to DNA damage or DNA replication stress by monoubiquitinating two substrates FANCD2 and FANCI (Fig. 1) (20, 78, 91, 129).

FIG. 1.

A simplified model of the Fanconi anemia (FA)/BRCA pathway. Eight FA proteins (FANCA, B, C, E, F, G, L, and M) along with FAAP100 and FAAP20 form a nuclear protein complex (FA core complex), which acts as ubiquitin ligase by monoubiquitinating two substrates FANCD2 and FANCI, followed by recruiting other downstream FA proteins, including FANCD1, FANCJ, and FANCN.

Ever since early research reported excessive oxygen toxicity in FA cells, a great number of studies have related FA to oxidative stress, which has been considered a critical factor in the pathogenesis of FA (1, 26, 28, 37, 52, 67, 69, 80, 84, 88, 90, 91, 113, 136, 137). Oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense, is associated with many human disease states, including FA, which is arguably a unique disease model characterized by abnormal accumulation of ROS and a dysfunctional response to oxidative damage (26, 91). Evidence has been accumulated that ROS, including hydrogen peroxide, superoxide, and the hydroxyl radical, could regulate the transduction of signals from the membrane to the nucleus via the modulation of cellular enzymatic activity by oxidation and reduction (98, 127). Among the enzymes modified by oxidative stress, protein kinases and phosphatases serve as important regulators of cellular signaling pathways (60, 98). Excessive generation of ROS has been shown to activate multiple protein kinases, such as extracellular signal-regulated kinase (ERK)1/2, protein kinase B (PKB), and protein tyrosine kinases (PTKs) (25, 31, 43, 50, 118, 125, 127). Additionally, a variety of phosphatases are the sensitive targets of oxidative stress and may be inactivated under oxidizing conditions, which would amplify the effect of redox-linked activation of key protein kinases (60, 110, 111, 131). Given that the balance between protein kinases and phosphatases dictates the overall phosphorylation state of cellular phosphoproteins, aberrant protein phosphorylation may exacerbate the pathophysiology of multiple human diseases and targeting them by small molecules therefore would be anticipated to ameliorate clinical symptoms (34, 38, 76, 79).

In this review, we will describe the abnormal protein kinases and phosphatases influenced by oxidative stress, in particular, aberrant protein tyrosine phosphatases (PTPs) in FA. Furthermore, we will discuss the involvement of abnormal protein phosphorylation during the development of clinical complications and the potential drug targets to improve the FA phenotype.

Impaired Tyrosine Phosphatases in Oxidative Stress

The reversible phosphorylation of protein residues, regulated by protein kinases and phosphatases, modulates many important aspects of cell life, including metabolism, transcription, cell cycle progression, cytoskeletal rearrangement, differentiation, and apoptosis (1, 13, 33, 111, 120). Protein phosphorylation status also plays a critical role in the intercellular communication during development, in physiological responses, and in homeostasis, and in the functioning of the nervous and immune systems (15, 24, 47, 81, 123). Impaired protein phosphorylation triggered by aberrant functions of protein kinases and phosphatases therefore contributes to a number of disorders (1, 13, 77, 86, 111). Protein kinases and phosphatases themselves may be direct targets of oxidative stress (1, 13, 40, 43, 77, 111, 117, 120). In particular, tyrosine phosphorylation of proteins controlled by PTKs and PTPs plays a key role in the pathogenesis of multiple disorders (63, 77, 93, 135). The regulation of PTPs is thus of vital importance for cell signaling.

The PTP superfamily is encoded by ∼100 genes in the human genome, which shares an overall structure with a core catalytic domain composed of four parallel β-strands surrounded by α-helixes on both sides (1, 7). The active site sequence Cys(X)5Arg is referred to as “PTP signature motif,” which connects a center β-strand to an α-helix at the center of the catalytic site. In addition, the diversity of PTPs is conferred by the regulatory domains (1, 13, 77, 85, 104, 117, 135). Furthermore, PTPs can be categorized by the sensitivity to vanadate, the insensitivity to okadaic acid, the ability to hydrolyze the ρ-nitophenyl phosphate, and lack of metal ion dependence for catalysis (7, 13). Functionally, PTPs can act both positively and negatively during the regulation of signal transduction through their specific dephosphorylation activity (120).

Based on phosphamino acid specificity, PTPs can be classified into four groups: the classical receptor (RPTPs), the classical nonreceptor PTP (nrPTPs), the dual specificity PTP (dsPTPs), and the low-molecular weight PTPs (low M_r PTPs) (1). Furthermore, RPTPs and nrPTPs fall into several subtypes according to their noncatalytic domain structures (1, 3, 95, 111, 120). RPTPs are predominantly found in the plasma membrane, whereas nrPTPs are localized to a variety of intracellular compartments, including the cytosol, plasma membrane, and endoplasmic reticulum (ER) (Fig. 2).

FIG. 2.

Schematic representation of the protein tyrosine phosphatase (PTP) family. The PTP family is broadly divided into non-transmembrane (nonreceptor, nrPTPs) and receptor-like PTPs (RPTPs), which can be further classified into subfamilies. RPTPs are transmembrane proteins consisting of an extracellular part and the intracellular domains. The extracellular part typically contains domains implicated in cell adhesion. The intracellular part consists of one PTP catalytic domain and, in some subfamilies, a PTP domain with little or no catalytic activity. nrPTPs only have a single PTP catalytically active domain, which is linked to domains that mediate protein–protein interactions, including Src-homology 2 (SH2).

A series of studies have indicated that the activities of PTPs can be regulated by a variety of mechanisms, including the binding of both agonistic or antagonistic ligands (RPTPs), dimerization followed by autophosphorylation (CD45 and PTP-α), and phosphorylation at tyrosine residues to provide the binding sites for Src-homology 2-containing proteins (PTP-α) (1, 111, 117). Increasing evidence indicated that PTP activities can also be regulated by the redox state within the cell (Fig. 3) (1, 40, 60, 111, 117). The catalytic site of the majority of PTPs contains a cysteine residue with pKa values in the range of 4–6, which makes these sites hypersusceptible to oxidation. It has been shown that a wide range of oxidants could interact with PTP-active sites to regulate the enzymatic activities. The redox regulation of tyrosine phosphatases depends on different oxidative stress conditions and is tissue specific, which frequently occurs within the catalytic site of the enzyme (13, 14).

FIG. 3.

Reactive oxygen species (ROS) mediate cell signaling to regulate the protein phosphatase in response to the various extracellular stimuli. Cellular ROS can be generated in response to multiple external stimuli, including ionizing radiation, H₂O₂, growth factors, cytokines, and ligands for G protein-coupled receptor (GPCR). As shown in the diagram, phosphatase may be inhibited by ROS via oxidization of cysteine residues in proteins to form cysteine sulfenic acid (Cys-SOH). Moreover, SHP-1 and SHP-2 activity could be inhibited by oxidative stress generated by ionizing radiation or low H₂O₂ concentrations in a Ca²⁺-dependent NOS and an S-nitrosylation-dependent manner. ROS levels also regulate the protein phosphorylation status through modulating the kinases and/or phosphatase reversibly.

Oxidation of the catalytic cysteine of PTPs occurs in vivo in response to intracellular ROS production, which may be stimulated by a variety of factors, including platelet-derived growth factor, epidermal growth factor, insulin, and several other cytokines and integrins (23, 63, 99, 119). Cysteine residues are also affected by extracellular ROS or by an imbalance of the redox potential within the cell (Fig. 3). Therefore, hydrogen peroxide or other radical species may oxidize cysteine residues in proteins to form cysteine sulfenic acid (Cys-SOH), which can be further stabilized by the formation of interior intramolecular disulfide (S-S) or sulfenyl-amide bonds. The last conformation is characterized by a five-member ring that is formed by the covalent linkage of an atom of the catalytic cysteine to amide nitrogen in a neighboring residue.

Most RPTPs contain two intracellular enzymatic domains (D1 and D2). Oxidative stress could inhibit RPTPs by changing the conformation (9, 66, 72). It was shown that H₂O₂ treatment induced a conformational change in the cytoplasmic region that was dependent on a cysteine residue in D2, which enhanced RPTP-α dimerization and inactivated phosphatase activity. Moreover, SHP-1 and SHP-2 activity could be inhibited by oxidative stress generated by ionizing radiation or low H₂O₂ concentrations in a Ca²⁺-dependent NOS and an S-nitrosylation-dependent manner (9, 66, 72). Further investigation indicated that oxidative stress-induced inhibition on PTP activity is reversible (46, 122, 131). ROS accumulation resulting from cytokine treatment decreases the PTP1B activity by oxidative inactivation, which can be restored by the treatment with NAC, an ROS scavenger (46, 57). H₂O₂ treatment also oxidized and inactivated several PTPs in vitro (63, 77, 135).

Oxidative Stress Contributes to the FA Cellular and Clinical Phenotype

Early studies by Nordenson and Joenje first reported excess oxygen toxicity in FA cells that accumulated oxidative DNA damage (52, 84). Nordenson revealed that spontaneous chromosomal instability in FA cells was mitigated by superoxide dismutase (SOD) (84). Joenje et al. later reported that chromosomal aberrations in FA were oxygen dependent (52). Since then, oxidative stress was found to modulate cell growth (26, 90, 91) and cycle in FA cells (26, 90–92, 105) and linked to FA-associated chromosomal instability (26, 52, 68, 90, 91, 100). Both in vitro and in vivo evidence indicated that FA cells were in a prooxidant state (1, 8, 11, 17, 18, 20–22, 26, 28, 37, 68, 69, 71–77, 87, 88, 90–92, 100, 103, 134). Exposure to hydrogen peroxide caused extra oxidative damage on both DNA and RNA in FA cells compared with the damage in normal cells (116). Excess luminol-dependent chemiluminescence (LDCL), a direct endpoint of ROS formation, was found in freshly drawn leucocytes from FA patients and, to a lesser yet significant extent, in FA heterozygotes (58). Pagano et al. observed that excessive accumulation of white blood cell 8-OHdG correlated both with LDCL and spontaneous chromosomal breaks, as well as increased oxidized glutathione (GSSG)/GSH ratio and plasma methyl-glyoxal levels (89). Altered production of the proinflammatory cytokine, tumor necrosis factor α (TNF-α) may also contribute to the prooxidant state in FA cells. It has been reported that TNF-α could enhance intracellular and extracellular O₂⁻ production and induce DNA breakage and cell death (8, 10, 32, 68, 112, 132). Moreover, in the presence of genotoxic agents, such as radiation or chemotherapeutic drugs, the cytotoxic effects of TNF-α can be potentiated. Overproduction of TNF-α in FA cells enhances intracellular events such as nuclear factor-κB (NF-κB) activation and transcriptional activity, and leads to constitutive activation of multiple signaling pathways, including mitogen-activated protein kinase (MAPK), NF-κB (two major stress-signaling pathways), and c-Jun NH2-terminal kinase (JNK) pathway (26, 88, 90, 105). Furthermore, TNF-α treatment upregulated the expression of FANCA and FANCG, and increased the FANCA/FANCG complex in the nucleus as well through the NF-κB pathway (71, 72) Indeed, TNF-α and its interactors have been implicated in FA pathogenesis (26, 88, 90, 105). Several FA gene products have been shown to play roles in redox homeostasis directly or indirectly (Fig. 4) (26, 37, 52, 67, 69, 90–92, 100, 105, 114, 116). Both FANCA and FANCG proteins were found to be directly influenced by the redox state in terms of physical structure (94). In response to the redox state, FANCA and FANCG proteins could form disulfide bonds in the FA protein complex (27, 94). The FANCC protein was reported to bind with NADPH cytochrome-P450 reductase and glutathione S-transferase P1-1 (37, 61). Futaki et al. revealed that FANCG interacts with a P450 protein, cytochrome P450 2E1 (CYP2E1) (35). Rani et al. reported that deficiency in the Fanca gene in mice elicits a p53-dependent growth arrest and DNA damage response to oxidative DNA damage and oncogenic stress (97). FANCD2 was found to interact with the ataxia-telangiectasia mutated protein (ATM) and forkhead box O3 (Foxo3a) in response to oxidative DNA damage (67), which is consistent with the established functions of both ATM and FOXO3a during oxidative stress (67, 121). FANCJ was recognized to coincide with the DNA helicase BRCA1-interacting protein 1 (BRIP1) and the transcription factor BRCA1-associated C-terminal helicase (BACH1) and that FANCJ/BACH1/BRIP1is a repressor of heme oxygenase-1(HO-1) gene and senses oxidative base damage (114).

FIG. 4.

Overall scenario of the relationships of FA proteins with oxidative stress-related interactors. Eight FA proteins form a nuclear complex, which acts as ubiquitin ligase by monoubiquitinating two substrates FANCD2 and FANCI, followed by recruiting other downstream FA proteins, including FANCD1, FANCJ, and FANCN. FA gene products involved in redox homeostasis can be summarized as follows: FA proteins and/or FA protein complexes and subcomplexes may interact with or regulate AKT, cyt450, nuclear factor-κB (NF-κB), GST, Cyp2E1; FANCJ is a repressor of heme oxygenase (HO)-1 gene and senses oxidative base damage; FANCG interacts directly with peroxyredoxin 3 (PRDX3) in mitochondria; FANCD2 in response to oxidative stress may interact with Foxo3a and ataxia-telangiectasia mutated protein (ATM).

Furthermore, some FA gene products correlated to oxygen metabolism are also associated with mitochondrial dysfunction. Mukhopadhyay et al. observed that FANCG localizes to mitochondria and interacts with mitochondrial peroxide, peroxyredoxin 3 (PRDX3) (80). In turn, PRDX3 was deregulated in FANCG-deficient cells, which may due to distorted mitochondrial structures, and mitochondrial extract showed significant decrease in thioredoxin-dependent peroxidase activity. Overexpression of PRDX3 overcomes the sensitivity of FA-G cells to hydrogen peroxide, and a decreased PRDX3 expression leads to hypersensitivity to mitomycin C. FANCA- and FANCC-deficient cells also had PRDX3 cleavage and decreased peroxidase activity (80, 102).

A set of murine FA models also link FA phenotypes to oxidative stress (42, 105, 136, 137). Hadjur et al. revealed defective hematopoiesis and hepatic steatosis in Fancc^−/−Sod1^−/− mice and the altered redox state was responsible for an impairment of cell proliferation or survival, and attributed to hepatocyte membranes (42). Sejas et al. found that Fancc^−/− mice exhibited excess inflammatory response as a result of hematopoietic suppression. The lipopolysaccharide-mediated hematopoietic suppression was elicited by TNF-α and triggered ROS formation, along with overexpression of the stress kinase p38 (105). Zhang et al. observed that long-term antioxidant administration could counteract FA-associated oxidative stress in Fancd2^−/− mice (136, 137). These studies demonstrated that Fancd2^−/− mice with tempol treatment, a nitroxide antioxidant and a SOD mimetic, showed a significant delay in the onset of epithelial tumors; and treatment with resveratrol improved the spleen colony-forming capacity of Fancd2^−/− bone marrow cells.

Altogether, these findings provide both direct and indirect evidence that oxidative stress is at least a component of FA cellular and clinical phenotype (Fig. 5) (5, 11, 17, 20, 26, 54, 68, 69, 88, 90, 94, 97, 129, 130).

FIG. 5.

Consequence of FA defects. The FA pathway serves during the DNA damaging sensing/signaling, DNA damage repair, and eliminating ROS by cooperating with various interactors, such as ATM/ATR, Foxo3a/STAT1, separately. Mutations of FA complementation groups lead to the FA phenotypes such as congenital abnormalities, cancer, and bone marrow failure.

Oxidative Stress-Associated Protein Phosphorylation and Dephosphorylation Abnormalities in FA

Oxidative stress due to excessive accumulation of ROS was reported to induce double-stranded RNA-dependent protein kinase (PKR)-dependent apoptosis via interferon-γ (IFN-γ) activation signaling (96). Pang et al. revealed that FANCC could bind to and facilitate the activation of STAT1 by IFN-γ and hematopoietic growth factors (92). Saadatzadeh et al. revealed that hyperactivation of ASK1 triggered by oxidative stress contributed to the H₂O₂- and TNF-α-induced apoptosis, which was ablated by ASK1-specific siRNA and a dominant negative ASK1 mutant to inhibit the ASK1 kinase activity (103). Further study from the Haneline group indicated that antioxidants or a p38 inhibitor could protect Fancc^−/− MEFs and c-Kit⁺ cells from TNF-α apoptosis by inhibiting ASK1 activity (8). Altogether, altered redox status may contribute to the apoptosis prone in FA cells by protein phosphorylation dysregulation (8, 92, 96, 103).

More direct evidence to elucidate the tyrosine kinases involved in the oxidative stress response or triggered by extra accumulation of ROS was obtained by human phospho-receptor tyrosine kinase (RTK) arrays reported by Li et al. (Fig. 6) (69). After H₂O₂ treatment, multiple tyrosine kinases, including insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), Janus kinase 2 (JAK2), anaplastic lymphoma kinase (ALK), TET tyrosine kinase, and endothelial (Tie 2), were activated differentially in human FA-C cells compared with the ones in normal human lymphoblastic cells, suggesting that FA deficiency impairs multiple tyrosine kinase signaling pathways.

FIG. 6.

Aberrant signaling triggered by H₂O₂ in FANCC-deficient patient-derived lymphoblastic cell compared with the one in human normal lymphoblastic cell. To identify tyrosine kinases involved in oxidative stress response in FA cells, human phospho-receptor tyrosine kinase (RTK) arrays were employed to screen 78 different RTKs for differentially activated kinases in response to oxidative stress induced by H₂O₂ treatment in human lymphoblastic cell lines (LCLs) derived from a normal donor (HSC93) (A, B) or a FA patient assigned to the complementation group C (FANCC; HSC536) (C, D). After H₂O₂ treatment, multiple tyrosine kinases, including insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), Janus kinase 2 (JAK2), anaplastic lymphoma kinase (ALK), TET tyrosine kinase, and endothelial (Tie 2), were activated differentially in human FANCC-deficient cells compared with the ones in normal human lymphoblastic cells.

Oxidative Stress-Associated Insulin Resistance in FA

One of the clinical characteristics of FA is the metabolic disorder, which is manifested by diabetes and other abnormalities of glucose metabolism (30, 39, 115). A recent clinical investigation showed that abnormalities of glucose homeostasis in FA patients were frequent (up to 81% of FA patients), including hyperglycemia (impaired glucose tolerance or diabetes mellitus) and hyperinsulinemia (30, 39, 115). However, the underlying molecular etiology of the FA diabetes remains unclear. Insulin resistance, a state that precedes many clinical manifestations of the metabolic syndrome by inhibiting the action of insulin, is a key contributor to the pathogenesis of obesity and type 2 diabetes (T2D) mellitus (2, 41). The literature indicated that mutations in the FANCA and FANCC genes among 15 complementation groups have been identified in more than 70% of FA patients worldwide (5, 21, 54, 74, 83, 100). We previously employed two FA knockout mouse models, Fanca^−/− and Fancc^−/−, to study the mechanisms contributing to the abnormalities of glucose homeostasis and insulin resistance in FA (Fig. 6) (69). Our results indicated that mice deficient for the Fanca or Fancc gene seem to be prone to diabetes and obesity, consistent with the clinical complication in FA patients. Specifically, both Fanca^−/− and Fancc^−/− mice showed higher fasting blood glucose and insulin resistance, as determined by the standard glucose tolerance test and insulin tolerance test (2, 30, 39, 69, 115).

Insulin signaling is critical for the regulation of glucose levels and the avoidance of diabetes mellitus, whereas defective insulin signaling may cause insulin resistance, leading to multiple diseases, including T2D and obesity (62, 128). The IR is activated through phosphorylation at multiple tyrosine residues of the β-subunit, which then phosphorylates and recruits different substrate adaptors, including members of the insulin receptor substrate (IRS) family and leads to the activation of downstream signaling, such as PI3K/AKT/PKB and PKCzeta cascades (12, 40, 124). Our results indicated that FA insulin resistance may have resulted from the reduction of IR tyrosine phosphorylation and an increase in the inhibitory serine phosphorylation of IRS-1 (Fig. 7) (69).

FIG. 7.

Schematic model for ROS-induced insulin resistance in FA. Overproduction of tumor necrosis factor α (TNF-α) leads to the accumulation of ROS and consequently influences two critical steps of IR signaling: decrease IR tyrosine phosphorylation through PTP-α; increase the inhibitory phosphorylation of insulin receptor substrate-1 (IRS-1) by activation of double-stranded RNA-dependent protein kinase (PKR) kinase, leading to insulin resistance. Inhibition of PTPalpa or PKR failed to restore glucose metabolism. The natural antioxidant Quercetin ameliorates the negative effect of ROS on insulin signaling and maintains metabolic homeostasis.

Numerous studies indicated that oxidative stress may contribute to the pathogenesis and clinical complications in FA. The IR signaling pathway is regulated tightly at multiple levels, including receptor phosphorylation and feedback of downstream signals, such as IRS-1, triggered by ROS (12, 44, 65, 82, 126, 127). Our investigation on the role of ROS in FA insulin resistance indicated that high levels of ROS contribute to the FA insulin resistance, which could be restored by the natural antioxidant quercetin, suggesting potential therapeutic treatment to improve the FA phenotype (69).

FA patients have abnormally high levels of TNF-α, which is a major mediator of inflammation and ROS production, leading to persistent PKR activation in FA mice (82, 91, 93, 101, 138). It was known that PKR phosphorylation induced by ROS could inhibit IRS-1 by phosphorylation at inhibitory site Ser312 in human liver cells HepG2 (IRS-1Ser307 in mouse cells) (82, 133). Consistent with the inhibitory effect of PKR on the insulin signaling triggered by oxidative stress, our inhibitor screen identified PKR kinase as one of the two factors that mediate the ROS effect of FA insulin resistance. Furthermore, using the shRNA screen, we identified the tyrosine phosphatase PTP-α as another critical factor that mediates the ROS effect on FA insulin resistance.

The identification of PKR kinase and PTP-α phosphatase that mediates the inhibitory effect of ROS on insulin signaling provides a potential mechanistic link between ROS and insulin resistance in FA. Given, targeting PKR or PTP-α alone failed to restore insulin signaling in FA cells, the amelioration of FA insulin resistance may require simultaneously targeting both molecular events, which can both restore IR tyrosine phosphorylation and suppress inhibitory IRS-1Ser307 phosphorylation. Indeed, the combined inhibition of both PKR and PTP-α rescues FA insulin resistance caused by TNF-α in FANCA knockdown cells (Fig. 7).

Proposal to Target PTP and PKR Kinase to Improve FA Phenotype

FA can cause a wide range of medical problems. There is no cure for this disease so far, however, many medical therapies, procedures, and surgeries can now treat the symptoms and improve the FA phenotype, including blood transfusions, blood and marrow stem cell transplant, androgen therapy, synthetic growth factors, and gene therapy (18, 19, 54, 134).

Scientific implication that oxidative stress may contribute to the pathogenesis and clinical complications in FA provides attractive therapeutic strategies by blocking the production of ROS or enhancing the DNA damage repair ability (70, 136, 137). In vivo experiments conducted with Fancd2^−/− mice revealed that antioxidants administered long-term could delay significantly the onset of epithelial tumors and improve the spleen colony-forming capacity by counteracting FA-associated oxidative stress (136, 137). Quercetin, a naturally occurring flavonoid found in a variety of fruits and green vegetables, has a wide range of biological activity such as free radical scavenging, iron chelating, anti-inflammation, and anticancer (56, 107). We have shown that quercetin effectively antagonized the negative effect of ROS on insulin signaling and maintaining metabolic homeostasis, and thus may be useful for therapies against insulin resistance-associated metabolic syndrome, as demonstrated in the FA disease model.

Redox abnormalities in FA may lead to abnormal activities of kinases and phosphatases, especially tyrosine kinases and phosphatases. Given the important roles of these enzymes in the modulation of cellular proliferation, differentiation, and viability, tyrosine kinases and phosphatases therefore become attractive and promising targets for therapies (Fig. 8) (4, 6, 29, 59, 64, 75, 86, 109). Tyrosine kinases can be inhibited pharmacologically through multiple mechanisms (Fig. 7). The small molecules may function through directly inhibiting the catalytic activity of the kinase by interfering with the binding of ATP or substrates, blocking the dimerization of fusion kinases or preventing the ligand binding (4, 6, 59, 75). The development of suitable inhibitors for PTPs is still under investigating although the emerging oncogenic function of members of the PTP family has led to their consideration as drug targets (29, 64, 86, 109). There are significant technical challenges for the PTP inhibitor design due to high polarity of the active compounds and the specificity of compounds to distinguish the closely related catalytic domains of functionally different PTPs (29, 64, 71, 73, 86, 109), and currently no specific PTP inhibitors have been clinically approved yet (45). Encouragingly, these problems have, in part, been addressed by the use of prodrug strategies and structural information techniques (29, 64, 71, 73, 86, 109).

FIG. 8.

PKR may serve as the therapeutic target in FA. FANCC associates with PKR and HSP70 (a PKR activation inhibitor) exposed to either combination of TNF-α and interferon-γ (IFN-γ) or dsRNA and IFN-γ. Constitutive activation of PKR in FA cells, caused by overproduced cytokines or accumulation of ROS due to FA complementation mutation, contributes to the FA clinical phenotype, including hypersensitivity to IFN-γ/TNF-α, which may be abrogated by PKR inhibition.

It has been shown that inhibition of PKR represents an interesting strategy for neuroprotection, and preventing the cell death induced by ER stress in cultured human neuroblastoma cells (16, 36, 48, 49, 51, 106, 139). PKR was constitutively activated in FA-C cells, which contributed to the hypersensitivity of bone marrow progenitor cells to growth repression mediated by the inhibitory cytokines IFN-γ and TNF-α (69, 92, 105, 138). Inhibition of PKR activation by overexpression of a nonphosphorylated form of eukaryotic translation initiation factor-α reversed PKR-mediated bock of messenger RNA translation and partly abrogated PKR-mediated apoptosis in response to IFN-γ, TNF-α, and dsRNA (69, 92, 105, 138). Further investigation indicated that overactivation of PKR may also trigger the inhibitory phosphorylation of IRS-1, which partly contributes to the insulin resistance and abnormal glucose metabolism (69). Therefore, it is reasonable to consider the PKR inhibitor as an interesting therapeutic candidate in FA patients. However, aberrant accumulation of cytokines in FA cells may trigger multiple signaling pathways besides PKR, particularly the bias caused by phosphatases may lead to an insufficient therapeutic benefit (69, 92, 105, 138).

Oxidative stress in FA leads to the aberrant phosphorylation status via disrupting the balance between kinases and phosphatases. Targeting phosphatase therefore may also serve as the potential therapeutic target to improve FA phenotype. PTP-α is reported to affect transformation and tumorigenesis, inhibition of proliferation and cell cycle arrest, neuronal differentiation and outgrowth. In FA, PTP-α also modulated insulin signaling through regulating the IR phosphorylation. Thus, PTP-α has promise as a target for drug discovery. However, inhibition of PTP-α alone may also fail to restore the functional insulin signaling, indicating that a combined therapeutic strategy may be an optimal way to improve FA phenotype.

Innovation

Hypersensitivity to oxidative stress has been considered to contribute to the syndrome progression in Fanconi anemia (FA). Recent studies indicated that redox status may cause the aberrant phosphorylation states of cellular proteins via regulating the balance between protein kinases and phosphatases, which may contribute to disease development, including FA. Current review summarized the latest understanding on the roles of FA proteins in modulating redox functions and highlighted the phosphorylation state of proteins, including protein tyrosine kinases and protein tyrosine phosphatases in FA cells, and provided a review of the evidence for molecular and clinical involvement of oxidative stress in the FA phenotype.

Abbreviations Used

ALK

anaplastic lymphoma kinase

ATM

ataxia-telangiectasia mutated protein

BACH1

BRCA1-associated C-terminal helicase

BRIP1

BRCA1-interacting protein 1

dsPTP

the dual specificity protein tyrosine phosphatase

ER

endoplasmic reticulum

ERK

extracellular signal-regulated kinase

FA

Fanconi anemia

FANCA

Fanconi anemia complementation group A

FANCC

Fanconi anemia complementation group C

FANCD2

Fanconi anemia complementation group D2

FANCG

Fanconi anemia complementation group G

FANCI

Fanconi anemia complementation group I

GPCR

G protein-coupled receptor

HO

heme oxygenase

IFN-γ

interferon-γ

IGF-1R

insulin-like growth factor 1 receptor

IR

insulin receptor

IRS

insulin receptor substrate

JAK2

Janus kinase 2

JNK

c-Jun NH2-terminal kinase

LDCL

luminol-dependent chemiluminescence

low M_r PTPs

low-molecular weight PTPs

MAPK

mitogen-activated protein kinase

NF-κB

nuclear factor-κB

nrPTP

the classical nonreceptor protein tyrosine phosphatase

PKB

protein kinase B

PKR

double-stranded RNA-dependent protein kinase

PRDX3

peroxyredoxin 3

PTK

protein tyrosine kinase

PTP

protein tyrosine phosphatase

ROS

reactive oxygen species

RPTP

receptor protein tyrosine phosphatase

RTK

receptor tyrosine kinase

SH2

Src-homology 2

T2D

type 2 diabetes

TNF

tumor necrosis factor

Acknowledgments

This work was supported, in part, by National Institutes of Health (grants R01 CA109641 and R01 HL076712). Q.P. is supported by a Leukemia & Lymphoma Scholar award.